This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A greater understanding of tympanic membrane (TM) biomechanics has the potential to guide future advances in medical technology related to the surgical repair of eardrums or myringoplasties. The TM is a composite structure with radial, circular, and parabolic arrangements of collagen fiber layers that provide the main scaffolding. A more detailed knowledge of collagen fiber orientation and volume fraction could greatly improve existing mechanical simulations of the TM. Human TM punch biopsies (1 mm diameter) were imaged by transmission electron microscopy (TEM). These samples were prepared using a newly developed method of ultra rapid high-pressure freezing and subsequent freeze substitution, which preserves ultra-structure better than comparable methods used previously in TEM studies of the TM. To guide our future studies of the TM, we will image the samples using Second Harmonic Generation microscopy (SHG). The SHG images will allow us to choose regions of interest to punch biopsy for subsequent TEM imaging. Our goal is to use the high-resolution (4 nm x 4 nm resolution) 2-D TEM images we have obtained to create 3-D tomographic images of pertinent regions. We will then integrate these smaller volume images into the 3-D reconstructions generated by the SHG imaging. We believe the three dimensional data will yield new insights into collagen fiber density and fiber orientation as a function of tympanic membrane thickness and radial position.